Probing substrate binding to Metallo-β-Lactamase L1 from Stenotrophomonas maltophilia by using site-directed mutagenesis

BACKGROUND: The metallo-β-lactamases are Zn(II)-containing enzymes that hydrolyze the β-lactam bond in penicillins, cephalosporins, and carbapenems and are involved in bacterial antibiotic resistance. There are at least 20 distinct organisms that produce a metallo-β-lactamase, and these enzymes have been extensively studied using X-ray crystallographic, computational, kinetic, and inhibition studies; however, much is still unknown about how substrates bind and the catalytic mechanism. In an effort to probe substrate binding to metallo-β-lactamase L1 from Stenotrophomonas maltophilia, nine site-directed mutants of L1 were prepared and characterized using metal analyses, CD spectroscopy, and pre-steady state and steady state kinetics. RESULTS: Site-directed mutations were generated of amino acids previously predicted to be important in substrate binding. Steady-state kinetic studies using the mutant enzymes and 9 different substrates demonstrated varying K(m) and k(cat) values for the different enzymes and substrates and that no direct correlation between K(m) and the effect of the mutation on substrate binding could be drawn. Stopped-flow fluorescence studies using nitrocefin as the substrate showed that only the S224D and Y228A mutants exhibited weaker nitrocefin binding. CONCLUSIONS: The data presented herein indicate that Ser224, Ile164, Phe158, Tyr228, and Asn233 are not essential for tight binding of substrate to metallo-β-lactamase L1. The results in this work also show that K(m) values are not reliable for showing substrate binding, and there is no correlation between substrate binding and the amount of reaction intermediate formed during the reaction. This work represents the first experimental testing of one of the computational models of the metallo-β-lactamases

Porphyromonas gingivalis initiates a mesenchymal-like transition through ZEB1 in gingival epithelial cells

The oral anaerobe Porphyromonas gingivalis is associated with the development of cancers including oral squamous cell carcinoma (OSCC). Here we show that infection of gingival epithelial cells with P. gingivalis induces expression and nuclear localization of the ZEB1 transcription factor which controls epithelial-mesenchymal transition (EMT). P. gingivalis also caused an increase in ZEB1 expression as a dual species community with Fusobacterium nucleatum or Streptococcus gordonii. Increased ZEB1 expression was associated with elevated ZEB1 promoter activity and did not require suppression of the miR-200 family of micro RNAs. P. gingivalis strains lacking the FimA fimbrial protein were attenuated in their ability to induce ZEB1 expression. ZEB1 levels correlated with an increase in expression of mesenchymal markers, including vimentin and MMP-9, and with enhanced migration of epithelial cells into matrigel. Knockdown of ZEB1 with siRNA prevented the P. gingivalis-induced increase in mesenchymal markers and epithelial cell migration. Oral infection of mice by P. gingivalis increased ZEB1 levels in gingival tissues, and intracellular P. gingivalis were detected by antibody staining in biopsy samples from OSCC. These findings indicate that FimA-driven ZEB1 expression could provide a mechanistic basis for a P. gingivalis contribution to OSCC

Characterization of monomeric L1 metallo-beta -lactamase and the role of the N-terminal extension in negative cooperativity and antibiotic hydrolysis

The L1 metallo-?-lactamase fromStenotrophomonas maltophilia is unique among this class of enzymes because it is tetrameric. Previous work predicted that the two regions of important intersubunit interaction were the residue Met-140 and the N-terminal extensions of each subunit. The N-terminal extension was also implicated in ?-lactam binding. Mutation of methionine 140 to aspartic acid results in a monomeric L1 ?-lactamase with a greatly altered substrate specificity profile. A 20-amino acid N-terminal deletion mutant enzyme (N-Del) could be isolated in a tetrameric form but demonstrated greatly reduced rates of ?-lactam hydrolysis and different substrate profiles compared with that of the parent enzyme. Specific site-directed mutations of individual N terminus residues were made (Y11S, W17S, and a double mutant L5A/L8A). All N-terminal mutant enzymes were tetramers and all showed higherK m values for ampicillin and nitrocefin, hydrolyzed ceftazidime poorly, and hydrolyzed imipenem more efficiently than ampicillin in contrast to wild-type L1. Nitrocefin turnover was significantly increased, probably because of an increased rate of breakdown of the intermediate species due to a lack of stabilizing forces. K m values for monomeric L1 were greatly increased for all antibiotics tested. A model of a highly mobile N-terminal extension in the monomeric enzyme is proposed to explain these findings. Tetrameric L1 shows negative cooperativity, which is not present in either the monomer or N-terminal deletion enzymes, suggesting that the cooperative effect is mediated via N-terminal intersubunit interactions. These data indicate that while the N terminus of L1 is not essential for ?-lactam hydrolysis, it is clearly important to its activity and substrate specificity

A Systems Biology Approach Identifies a Regulatory Network in Parotid Acinar Cell Terminal Differentiation

<div><p>Objective</p><p>The transcription factor networks that drive parotid salivary gland progenitor cells to terminally differentiate, remain largely unknown and are vital to understanding the regeneration process.</p><p>Methodology</p><p>A systems biology approach was taken to measure mRNA and microRNA expression in vivo across acinar cell terminal differentiation in the rat parotid salivary gland. Laser capture microdissection (LCM) was used to specifically isolate acinar cell RNA at times spanning the month-long period of parotid differentiation.</p><p>Results</p><p>Clustering of microarray measurements suggests that expression occurs in four stages. mRNA expression patterns suggest a novel role for <i>Pparg</i> which is transiently increased during mid postnatal differentiation in concert with several target gene mRNAs. 79 microRNAs are significantly differentially expressed across time. Profiles of statistically significant changes of mRNA expression, combined with reciprocal correlations of microRNAs and their target mRNAs, suggest a putative network involving <i>Klf4</i>, a differentiation inhibiting transcription factor, which decreases as several targeting microRNAs increase late in differentiation. The network suggests a molecular switch (involving <i>Prdm1</i>, <i>Sox11</i>, <i>Pax5</i>, miR-200a, and miR-30a) progressively decreases repression of <i>Xbp1</i> gene transcription, in concert with decreased translational repression by miR-214. <i>The transcription factor Xbp1</i> mRNA is initially low, increases progressively, and may be maintained by a positive feedback loop with <i>Atf6</i>. Transfection studies show that <i>Xbp1Mist1</i> promoter. In addition, <i>Xbp1</i> and <i>Mist1</i> each activate the parotid secretory protein (<i>Psp</i>) gene, which encodes an abundant salivary protein, and is a marker of terminal differentiation.</p><p>Conclusion</p><p>This study identifies novel expression patterns of <i>Pparg</i>, <i>Klf4</i>, and <i>Sox11</i> during parotid acinar cell differentiation, as well as numerous differentially expressed microRNAs. Network analysis identifies a novel stemness arm, a genetic switch involving transcription factors and microRNAs, and transition to an <i>Xbp1</i> driven differentiation network. This proposed network suggests key regulatory interactions in parotid gland terminal differentiation.</p></div

<i>Xbp1</i> Regulates <i>Mist1</i> Expression during Parotid Differentiation.

<p>(A) Log2 plot of microarray data for <i>Xbp1</i> and <i>Mist1</i>. (B) Expression of <i>Xbp1</i> and <i>Mist1</i> is highly correlated across parotid differentiation. Plot of Log2 <i>Xbp1</i> vs. Log2 <i>Mist1</i> shows a linear trend with R² = 0.9538. (C) Luciferase assay shows activation of <i>Mist1</i> promoter by <i>Xbp1</i> in ParC5 cells. Increasing amount of <i>Xbp1</i>-S (spliced <i>Xbp1</i>) cDNA/well (0.25 μg, 0.5 μg, and 1 μg) were co-transfected with a luciferase expression plasmid driven by a <i>Mist1</i> promoter. Significant increase in luciferase expression was observed for all concentrations of <i>Xbp1</i>-S (p = 0.017, p = 0.01, and p = 0.05 respectively) (n = 3).</p

<i>Psp</i> is Directly Regulated by both <i>Xbp1</i> and <i>Mist1</i>.

<p>(A) <i>Xbp1</i> activates the <i>Psp</i> promoter. Increasing amounts of <i>Xbp1</i>-S cDNA was co-transfected into ParC5 cells along with a luciferase expression plasmid driven by either a 500 bp or 1 kb fragment of the <i>Psp</i> promoter region. Analysis was performed by t-test. Expression of luciferase driven by 1 kb <i>Psp</i> promoter increases significantly upon increasing transfection of <i>Xbp1</i>-S (p = 0.007, 0.02, and 0.005 respectively). The same is seen with the 500bp <i>Psp</i> promoter (p = 0.01, 0.01, and 0.003 respectively) (n = 3). (B) <i>Mist1</i> activates the <i>Psp</i> promoter through interactions with intronic sequences. Luciferase expression was driven by either a 1.5 kb fragment of the <i>Psp</i> promoter or the 1.5 kb fragment along with 1320 bp of intronic sequence flanking exon 3 that contains two E-boxes. Promoter plasmids were co-transfected with <i>Mist1</i> and <i>Tcf3</i> cDNA expression plasmids. Analysis was performed by t-test (p = 0.02) (n = 4).</p

Transient Activation of <i>Pparg</i> during Parotid Acinar Cell Differentiation.

<p>(A) Network showing transcription factor <i>Pparg</i> and known downstream target genes found in DE Cluster 4 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125153#pone.0125153.g002" target="_blank">Fig 2</a>). DE Cluster 4 contains 106 genes (including <i>Pparg</i>) with a unique expression pattern; higher expression only in stages 2 and 3. The Metacore knowledge-base identifies 18 of these as <i>Pparg</i> target genes. A green arrow indicates activation of transcription while red arrow indicates inhibition. A grey line means the interaction is uncharacterized. Although a red arrow connects <i>Pparg</i> and <i>ACP5</i>, some publications list the interaction as activating [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125153#pone.0125153.ref067" target="_blank">67</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125153#pone.0125153.ref068" target="_blank">68</a>] indicating it could be context dependent. (B) Log2 expression of <i>Pparg</i> from microarray data. (C) qPCR data confirming the expression profile of <i>Pparg</i>. RNA samples from independent animals were collected at three time points (E20, P5, and P25). Expression was normalized to <i>Arbp</i>, and data showed significant change in expression by ANOVA. n = 3.</p